Device for measuring light emitted by microscopically small particles or biological cells

ABSTRACT

With a device for measuring the fluorescent light or scattered light emitted by microscopically small particles or cells ( 12 ), with a flow-through cuvette ( 8 ) through which the luminescent particles or cells ( 12 ) are guided, whereby the flow-through cuvette ( 8 ) has a transparent window ( 3 ), and with a photodetector ( 2 ), which records the light emitted by the luminescent particles or cells ( 12 ), and with an optical element that guides the light emerging from the window ( 3 ) to the photodetector ( 2 ), the invention suggests that the optical element is embodied as a cylinder ( 4, 9 ) with a cylindrical reflecting surface.

Methods are known for the characterization of microscopically smallparticles or biological cells, in which methods these particles areguided in suspension through an intense light beam, e.g., of a laser.The light scattered by the particles is received at different angles andrecorded and measured by means of sensitive photodetectors. In order todifferentiate between different cell types, the cells are marked bymeans of special fluorescent dyes. Different cells can be stained withdifferent markers. If these cells pass the light beam for fluorescenceexcitation, they emit fluorescent light that is recorded and quantifiedlikewise by means of photodetectors. In this manner it is possible todifferentiate from one another and count, e.g., different blood cells orleucocytes in a single measuring process.

This method known under the term “flow cytophotometry” or “flowcytometry” has become widely used in medical diagnostics. It serves,e.g., for automated cancer cell recognition, the quantification ofleucocyte subpopulations and the evaluation of the immune status ofHIV/AIDS patients by recording and counting the leucocytes responsiblefor immune defense. In addition, these methods serve as analyticalmethods using microscopically small plastic particles to which thebiochemical substances to be measured, such as nucleic acids andproteins, are bonded together with fluorescent dyes. The use in thecontext of flow cytophotometry is mentioned below in a simplified mannerand representing other possible applications.

The metrological task is to effectively record the very small amounts oflight emitted by cells or particles of such smallness. The cells orparticles move through the light beam very rapidly, so that it ispossible to measure as many of them as possible within a short time.Modern flow cytometers record more than 10,000 individual particles persecond with dwell times in the light beam of below 10 μsec. In terms ofmetrology this means that the integration times for the lightmeasurement are very short.

It is rendered possible to ensure a sufficient measuring sensitivityanyway in that the light emitted by the particles is recorded bymicroscope lenses that have a high numerical aperture, so that a highshare of the light is recorded. Optical elements are thus used between aflow-through cuvette and a photodetector, which elements are to supplyto the photodetector the highest possible share of the light emitted bythe cells. All flow cytophotometers known in practice use similar lenseswith high numerical aperture to record the largest possible solid angleand thus the highest possible share of the fluorescent light emitted bythe cells, since the light is not reflected by the particles in aparticular direction, but released in a spherical manner. Thefluorescent light reaches the photoelectric receiver or severalphotoelectric receivers via optical elements, lens systems andintermediate images arranged downstream.

It is characteristic of these known optical arrangements that theoptical systems used require a very precise optical image of themeasuring point or the cell to be measured because of the low focusdepth associated with the high numerical aperture. This leads to highdemands on the precision of the components, the mechanical stability ofthe entire measuring arrangement and thus to a considerable adjustmentcomplexity. The correspondingly high sensitivity of these measuringsystems does not allow, e.g., mobile use, e.g., in mobile laboratoriesfor the microbiological monitoring of bodies of water or for determiningas a part of therapy the immune status of HIV/AIDS patients in regionswithout any basic medical care or without laboratory infrastructure.

The object of the invention is to improve a generic device by embodyingit to be rugged while maintaining measuring sensitivity and measuringprecision and by making it suitable for use in mobile laboratories, andby making it possible for the maintenance expenditure, the amount ofadjustment interventions and the susceptibility to damage to be reducedor completely eliminated.

This object is attained by a device with the features of claim 1.

In other words, the invention suggests embodying the optical element asa cylinder with a cylindrical reflecting surface. Instead of acomplicated structure containing several optical lenses, a rugged,one-piece optical element is used that does not require any adjustmentor maintenance at all. Within the scope of the present suggestion,cylinder thereby means, as explained below, both solid and also hollowbodies. The cross-sectional shape of a cylinder of this type ispreferably circular, e.g., because of manufacturing advantages; but theadvantages according to the suggestion also arise with cross sectionsshaped in a manner deviating from a circle, e.g., ovaloid or polygonalcross sections. The cylindrical reflecting surfaces produce a tunnel forthe light through which it goes from the flow-through cuvette to thephotodetector virtually without any loss.

Apart from an improved ruggedness, the suggested embodiment of thedevice renders possible further advantages:

Improved Sensitivity of the Device:

The suggested arrangement allows the measurement of the smallest amountsof light emitted by microscopically small particles or cells. Comparedto the prior art, this new arrangement does not require any lenses orcomplicated lens systems. The disadvantages of the lenses with highnumerical aperture otherwise required and the high adjustment complexityassociated therewith are thus eliminated. The suggested arrangementrequires considerably fewer optical interfaces. This causes fewer lightlosses and thus greater measuring sensitivity.

A correspondingly large diameter of the light-collecting cylinder makesit possible to ensure that the share of light that can be recordedmetrologically at all reaches the light-collecting cylinder, i.e., thespace enclosed by the cylindrical reflecting surface, as completely aspossible. This is linked to the fact that the solid angle of thefluorescent light to be recorded is essentially determined by the totalreflection angle at the interfaces of liquid and glass wall in thecuvette channel and cuvette window. Only the share of the sphericallyemitted fluorescent light emitted within this solid angle can go fromthe flow-through cuvette to the photodetector. Purely by way of example,the diameter of the cylindrical reflecting surface can be approximately50 times bigger or even larger than the minimum distance between thelight-emitting particle and the light entrance opening of the cylinder.

Compared to the prior art, a further advantage of the suggestedarrangement is the fact that a larger solid angle and thus a greatershare of the fluorescent light emitted is recorded than is possible witha conventional optical lens system. If the cylinder is embodied, e.g.,not as a hollow cylinder, but as a solid body, and if it is made ofglass, it is connected to the flow-through cuvette or to its windowpreferably by means of an optical putty. The solid angle of the recordedlight is thus enlarged considerably again. This is because there arefewer losses through reflection at interfaces, which losses cannot beavoided with conventional lens options.

Simpler Assembly and Startup of the Device:

Compared to an optical lens system, first, the number of the componentsrequired is reduced so that, first of all, the production of the deviceis simplified in principle.

Secondly, if the diameter of the cylindrical reflecting surface isenlarged even more than stated above, it is possible for the share oflight corresponding to the greatest possible solid angle to reach thelight-collecting cylinder as completely as possible, even if theparticle to be measured is not located optimally close to the lightentrance opening of the cylinder, so that the distance from thelight-collecting cylinder is noncritical, similar to an enlarged focusdepth range of an optical lens system. Advantageously, the diameter ofthe cylindrical reflecting surface of the cylinder is thus comparativelylarge. As a result, the position of the particle to be measuredperpendicular to the observation direction is noncritical as well,namely advantageously over the entire width of the cuvette channel, sothat all the light-emitting particles or cells can reliably be recordedmetrologically with maximum luminous efficacy. Irrespective of theposition of the particle, it is always the same solid angle of thefluorescent light, i.e., the same share of the fluorescent lightradiated spherically overall, that reaches the light-collecting cylinderor tunnel. The sensitive adjustment of the measuring cell with respectto the photodetector can thus be omitted.

Simple Operability of the Device:

The suggested device for the light recording of microscopic particlesdoes not require readjustment or complex optical adjustment, namelyneither during setup nor later on during operation. All the componentsare permanently connected to one another in a fixed manner. As a result,the arrangement is insensitive to vibrations and sufficiently rugged torender possible use in mobile laboratories. The suggested device thusopens up completely new fields of application of flow cytophotometry, inwhich fields the arrangements hitherto known could not be used becauseof their sensitivity. For instance, an immunodiagnosis accompanying thetherapy of HIV/AIDS patients in rural areas of the poorest countries isthus rendered possible.

In particular if, as mentioned previously, the diameter of the cylinderis large, the advantages of a so-called “high numerical aperture” arise,as are known from optical lens systems in principle, but are notrealized with generic devices, and as cannot be achieved with customaryso-called optical waveguides made of optical fibers, since thesewaveguides have a considerably lower numerical aperture.

Exemplary embodiments of the invention are explained in more detailbelow on the basis of the purely diagrammatical drawings. They show

FIG. 1 A first exemplary embodiment with a measuring device featuring ahollow cylinder,

FIG. 2 A second exemplary embodiment, in which two measuring devicessimilar to that of FIG. 1 are provided, and

FIG. 3 A third exemplary embodiment with a measuring device featuring asolid cylinder.

The drawings always represent a highly schematized section of a flowcytophotometer. FIG. 1 shows that two partial flows (respectivelyindicated by arrows) of a transport fluid 10 merge in a manner known perse above a tubule 11. Fluorescently marked cells 12 to be counted emergeindividually at the top end of the tubule 11 and are transported throughan interior channel 1 of a flow-through cuvette 8 of the device togetherwith the transport fluid 10. There they are irradiated by a thin laserbeam and excited to emit fluorescent light. In the drawing, the laserbeam runs perpendicular to the drawing plane; it is thereforediscernible merely as a point impinging on a cell 12 and marked by anarrow labeled L. The width of the interior channel 1 discernible fromFIG. 1 is so small that the cells 12 enter into the laser beamindividually and create individual, distinguishable and thus countablelight pulses.

The interior channel 1 is closed off by a glass wall in the form of athin-walled window 3 in the direction of a photon receiver orphotodetector 2. Typically, the wall thickness is 0.2 to 1 mm. A hollowcylinder 4 with an interior diameter of typically 8 to 10 mm, a diameterthat is large compared to the width of the interior channel 1, rests onthis window 3. The interior wall surface of the hollow cylinder 4 ismirrored, so that a cylindrical reflecting surface for the fluorescentlight entering the hollow cylinder 4 is the result. In favor of theruggedness of the entire device, the hollow cylinder 4 can be made ofmetal, but other materials are possible as well, whereby those materialsare preferably used that render possible a very smooth surface.Depending on the material used, its interior wall surface can bepolished, so that the material of the hollow cylinder 4 directly formsthe cylindrical reflecting surface. In the exemplary embodiment shown,however, the interior wall surface is provided with an additionalreflective layer 5 having optimal reflecting properties, which layercreates the cylindrical reflecting surface.

In the direction of the photodetector 2, a fluorescent light filter 6 isconnected to the hollow cylinder 4, which filter allows the fluorescentlight to pass that is to be measured by the cells. The photodetector 2is located behind the fluorescent light filter 6. On the side of theflow-through cuvette 8 opposite the photodetector 2, the cuvette windowis provided with a reflective layer 7. The efficacy of the lightcollected is thus increased.

With a further exemplary embodiment according to FIG. 2, an independentmeasuring device is respectively arranged on both sides of theflow-through cuvette 8, which devices are both embodied according toFIG. 1: correspondingly, they have two internally mirrored hollowcylinders 4, fluorescent light filters 6 and photodetectors 2. By usingtwo different fluorescent light filters 6, different light wavelengthscan thus be recorded in a single measuring step.

In a further exemplary embodiment according to FIG. 3 it is providedthat the cylindrical reflecting surface is not formed by an internallymirrored hollow cylinder, but by a solid cylinder 9 in the form of aglass rod that is mirrored externally, and the exterior diameter ofwhich corresponds to the interior diameter of the hollow cylinder 4 ofFIG. 1. The reflective layer 5, applied externally in this case, formsthe cylindrical reflecting surface and can be provided with a protectivelayer (not shown for reasons of clarity), e.g., a protective varnish,which protects the reflective layer from atmospheric exposure. Similarto the exemplary embodiment of FIG. 2, the arrangement of two measuringdevices can also be provided with a device according to FIG. 3, whichmeasuring devices in this case contain solid cylinders 9, respectively.

It is respectively provided with all of the devices to protect themeasuring devices and in particular the cylinders 4 and 9 frommechanical influences by a housing (not shown in the drawings).

FIG. 1 indicates a solid angle 14 of the fluorescent light to berecorded. This solid angle 14 is essentially determined by the totalreflection angle at the interfaces of liquid and glass wall in theinterior channel 1 and window 3. As a result, the distance of theparticle to be measured from the light-collecting hollow cylinder 4 isnoncritical. The mirrored light-collecting hollow cylinder 4 has acomparatively large diameter. As a result, the position of the particleto be measured perpendicular to the observation direction is noncriticalas well. Irrespective of the position of the particle, the light fromthe same solid angle 14 of the fluorescent light always reaches thehollow cylinder 4.

1. Device for measuring the fluorescent light or scattered light emittedby microscopically small particles or cells (12), with a flow-throughcuvette (8) through which the luminescent particles or cells (12) areguided, whereby the flow-through cuvette (8) has a transparent window(3), and with a photodetector (2), which records the light emitted bythe luminescent particles or cells (12), and with an optical elementthat guides the light emerging from the window (3) to the photodetector(2), characterized in that the optical element is embodied as a cylinder(4, 9) with a cylindrical reflecting surface.
 2. Device according toclaim 1, characterized in that the cylinder is embodied as a hollowcylinder (4), the interior surface of which forms the cylindricalreflecting surface.
 3. Device according to claim 1, characterized inthat the cylinder is embodied as a solid, transparent cylinder (9), theexterior surface of which forms the cylindrical reflecting surface. 4.Device according to claim 1, characterized in that a fluorescent lightfilter (6) transparent to fluorescent light is located between theflow-through cuvette (8) and the photodetector (2).
 5. Device accordingto claim 1, characterized in that an independent light-measuringarrangement is respectively located on two sides of the flow-throughcuvette (8), which arrangements are equipped with different filterstransparent merely to fluorescent light.
 6. Device according to claim 4,characterized in that fluorescent light filters (6) are arranged on bothsides of the flow-through cuvette (8), whereby the two fluorescent lightfilters (6) are transparent to different light wavelengths.
 7. Deviceaccording to claim 1, characterized in that the cylindrical reflectingsurface of the cylinder (4, 9) has a circular cross section.
 8. Deviceaccording to claim 1, characterized in that the cylinder (4, 9) has ahigh numerical aperture.
 9. Device according to claim 8, characterizedin that the diameter of the cylindrical reflecting surface is at leastbig enough to allow the share of light emitted by a particle or a cell(12) within the maximum solid angle (14) theoretically possible andlimited by the total reflection in the area of the flow-through cuvette(8), to reach as completely as possible the interior space enclosed bythe cylindrical reflecting surface.
 10. Device according to claim 1,characterized in that the cylindrical reflecting surface is formed by acoating of the cylinder (4, 9).